P2Y6 receptor agonists for treating lung diseases

ABSTRACT

This invention is directed to a method of enhancing or facilitating the clearance of the lung mucus secretions in a subject. This invention is also directed to a method of facilitating the hydration of the lung mucus secretions in a subject. This invention is further directed to a method of preventing or treating diseases or conditions associated with impaired lung or airway function in a human or other mammal. The method comprises administering to a subject a pharmaceutical composition comprising a therapeutic effective amount of P2Y 6  receptor agonist compound, wherein said amount is effective to activate the P2Y 6  receptors on the luminal surface of lung epithelia. The P2Y 6  receptor agonist compounds useful for this invention include mononucleoside 5′-diphosphates, dinucleoside monophosphate, dinucleoside diphosphates, or dinucleoside triphosphates of general Formula I, or salts, solvates, hydrates thereof.

This application claims priority to U.S. provisional application No.60/695,358, filed Jun. 29, 2005. The content of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

This invention relates to P2Y₆ receptor agonists compounds and themethod of using such compounds in the treatment of diseases orconditions associated with impaired lung and airway function in humansand other mammals.

BACKGROUND OF THE INVENTION

The P2 receptor subtypes P2Y₂, P2Y₄, P2Y₆ and the adenosine receptorsubtype A_(2B) are all expressed in human airways. Instillation of ATP,UTP, or UDP into the lungs produces an increase in the mucociliaryclearance process in the airway epithelium. This response toextracellular nucleotides is produced by a number of events triggered byactivation of one or more P2Y receptors, resulting in an increase influid and chloride secretion (P2Y₂, P2Y₄, and P2Y₆), inhibition ofsodium absorption (P2Y₂, P2Y₄, and P2Y₆), and increase in mucinsecretion (P2Y₂ and P2Y₄). These events, in concert with an increase inciliary beat frequency, are responsible for the natural clearance of theairways. See Kellerman, Chest. 121:201S-205S (2002); Knowles, J ClinInvest. 109:571-7 (2002); Leipziger, Am J Physiol Renal Physiol. 284:F419-32 (2003); Kunzelmann, Clin Exp Pharmacol Physiol. 28:857-67(2001);and Schwiebert, Biochim Biophys Acta. 1615:7-32 (2003).

The role of P2Y₆ receptors has not been as clearly defined as that ofP2Y₂ receptors and their agonists due in part to the lack of selectiveP2Y₆ agonists. Stimulation of P2Y₆ receptors causes a dose-dependentincrease of fluid secretion, chloride secretion and ciliary beatfrequency (Morse, Am. J. Physiol. Cell Physiol. 280:C1485-497 (2001)).UDP, the natural agonist of P2Y₆ receptors, has lower efficacy promotingmucociliary clearance than agonists of P2Y₂ receptors, whether the lowefficacy of the natural P2Y₆ receptor agonist UDP is due to itsmetabolic liability is not known.

Mucus clearance is the cornerstone in the innate defense mechanism ofthe lung against disease produced by inhaled bacteria, viruses,chemicals and particulate material. In several lung diseases such aschronic obstructive pulmonary diseases (COPD), mucus hypersecretion,decreased clearance and inflammation contribute to airflow obstruction,resulting in destructive changes in the pulmonary parenchyma associatedwith an increase of morbidity and mortality. In these patients, airwayclearance therapy is necessary to prevent infection, increaseoxygenation and prevent progression of the decline in lung function.

SUMMARY OF THE INVENTION

This invention is directed to a method of enhancing or facilitating theclearance of the lung mucus secretions of a subject in need of suchtreatment. This invention is also directed to a method of facilitatingthe hydration of lung mucus secretion of a subject in need of suchtreatment. This invention is further directed to a method of preventingor treating diseases or conditions associated with impaired lung orairway function in a human or other mammal. Such diseases includechronic obstructive pulmonary disease (COPD), chronic bronchitis,emphysema, cystic fibrosis, primary ciliary dyskinesia (PCD), and alpha1 antitrypsin deficiency.

The methods comprise administering to a subject a therapeutic effectiveamount of a selective P2Y₆ receptor agonist compound, wherein saidamount is effective to activate the P2Y₆ receptors on the luminalsurface of lung epithelia. The P2Y₆ receptor agonist compound, forexample, is administrated to a subject by inhalation.

The P2Y₆ receptor agonist compounds useful for this invention areselective for P2Y₆ receptors, and include mononucleoside5′-diphosphates, dinucleoside monophosphate, dinucleoside diphosphates,or dinucleoside triphosphates of general Formula I, or salts, solvates,hydrates thereof. The present invention further provides pharmaceuticalformulations comprising a pharmaceutical carrier and a compound ofgeneral Formula I.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

When present, unless otherwise specified, the following terms aregenerally defined as, but are not limited to, the following:

Alkyl groups are from 1 to 12 carbons inclusively, either straightchained or branched, with or without heteroatoms, are more preferablyfrom 1 to 8 carbons inclusively, and most preferably 1 to 6 carbonsinclusively.

Alkenyl groups are from 1 to 12 carbons inclusively, either straight orbranched containing at least one double bond but may contain more thanone double bond, with or without heteroatoms.

Alkynyl groups are from 1 to 12 carbons inclusively, either straight orbranched containing at least one triple bond but may contain more thanone triple bond, and additionally may contain one or more double bondedmoieties, with or without heteroatoms.

Cycloalkyl groups are from 3 to 12 carbons inclusively, more preferablyfrom 3 to 10 carbons inclusively, and most preferably 3 to 6 carbonsinclusively, with or without heteroatoms.

Cycloalkenyl groups are from 4 to 12 carbons inclusively containing atleast one double bond, and with or without heteroatoms.

Aralkyl groups are from 1 to 8 carbons inclusively in the alkyl portion,are more preferably from 1 to 6 carbons inclusively in the alkylportion, and most preferably are 1 to 4 carbons inclusively in the alkylportion; in addition to the alkyl definition above, the alkyl portion ofan aralkyl group can include one or more positions of unsaturation suchas a double bond or a triple bond in the chain when the chain includestwo or more carbon atoms; the alkyl portion of an aralkyl group can alsoinclude one or more heteroatoms and/or substituents; the aryl portion ofan aralkyl group can be a monocyclic or polycyclic moiety from 3 to 8carbons inclusively per ring in the aryl portion, more preferably from 4to 6 carbons inclusively per ring, and most preferably 5 to 6 carbonsinclusively per ring; the aryl portion of an aralkyl group can also bearone or more substituents and/or heteroatoms.

Aryl groups are either monocyclic or polycyclic, are from 3 to 8 carbonsinclusively per ring, are more preferably from 4 to 6 carbonsinclusively per ring, and are most preferably 5 to 6 carbons inclusivelyper ring; aryl groups can also bear substituents and/or heteroatoms.

Heteroaralkyl groups are from 1 to 8 carbons inclusively in the alkylportion, are more preferably from 1 to 6 carbons inclusively in thealkyl portion, and most preferably are 1 to 4 carbons inclusively in thealkyl portion; in addition to the alkyl definition above, the alkylportion of a heteroaralkyl group can include one or more positions ofunsaturation such as a double bond or a triple bond in the chain whenthe chain includes two or more carbon atoms; the alkyl portion of aheteroaralkyl group can also include one or more heteroatoms and/orsubstituents; the heteroaryl portion of a heteroaralkyl group can be amonocyclic or polycyclic moiety from 3 to 8 carbons inclusively per ringin the heteroaryl portion and containing from 1 to 4 heteroatomsinclusively per ring, more preferably from 4 to 6 carbons inclusivelyper ring, and most preferably 5 to 6 carbons inclusively per ring; theheteroaryl portion of an heteroaralkyl group can also bear one or moresubstituents and/or heteroatoms.

Heteroaryl groups are either monocyclic or polycyclic, contain from 1 to4 heteroatoms inclusively per ring, are from 3 to 8 atoms inclusivelyper ring, are more preferably from 4 to 6 atoms inclusively per ring,and are most preferably 5 to 6 atoms inclusively per ring; heteroarylgroups can also bear substituents and/or heteroatoms.

Substituents on the foregoing groups can be, but are not limited to,hydroxy, nitro, methoxy, fluoro, chloro, bromo, iodo, methyl, ethyl,propyl, butyl, thioalkyl, alkoxy, carboxyl, carboxamido, alkylsulfonyl,alkylsulfonylamino, sulfonamido, cyano, amino, substituted amino,trifluoromethyl, trifluoromethoxy, phenyl, pyridyl, imidazolyl,cyclopropyl, cyclopentyl, and cyclohexyl; and preferred heteroatoms areoxygen, nitrogen, and sulfur.

A desired substituent on a chain or ring (in place of a hydrogen at aposition) is one selected from the given alkyl, aryl, halogen, aralkyl,carboxy, alkoxycarbonyl, hydroxyl, acyloxy, alkoxy, aryloxy or aralkoxyclasses or from other classes, which provides a compound withgood-to-excellent P2Y₁₂ receptor-binding properties, but which does notyield a compound with undesirable properties like chemical instabilityin a formulation, or one with levels of toxicity that are notwell-tolerated by a treated mammal, or especially, not well-tolerated bya human.

Diastereomers are stereoisomers (isomers of identical constitution butdiffering three-dimensional architecture), which do not bear amirror-image relation to each other.

Pharmaceutically acceptable salts are salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects. Pharmaceutically acceptable salt forms includevarious polymorphs as well as the amorphous form of the different saltsderived from acid or base additions. The acid addition salts can beformed with inorganic or organic acids. Illustrative but not restrictiveexamples of such acids include hydrochloric, hydrobromic, sulfuric,phosphoric, citric, acetic, propionic, benzoic, napthoic, oxalic,succinic, maleic, malic, mesylic, adipic, lactic, tartaric, salicylic,methanesulfonic, 2-hydroxyethanesulfonic, toluenesulfonic,benzenesulfonic, camphorsulfonic, and ethanesulfonic acids. Thepharmaceutically acceptable base addition salts can be formed with metalor organic counterions and include, but are not limited to, alkali metalsalts such as sodium or potassium; alkaline earth metal salts such asmagnesium or calcium; and ammonium or tetraalkyl ammonium salts, i.e.,NX₄ ⁺ (wherein X is C₁₋₄). Other salts such as hydrochlorides,hydrobromides, mesylates, sulfates, acetates, tartrates, etc., are alsocontemplated in this invention. Preferred counterions are monovalentions such as NH₄ ⁺, sodium, lithium, potassium, chloride, bromide,bisulfate, and mesylate, with sodium, potassium, chloride and mesylatebeing most preferred due to ease of manufacture, stability, andphysiological tolerance.

Solvates are addition complexes in which a compound is combined with apharmaceutically acceptable cosolvent in some fixed proportion.Cosolvents include, but are not limited to, water, methanol, ethanol,1-propanol, isopropanol, 1-butanol, isobutanol, tert-butanol, acetone,methyl ethyl ketone, acetonitrile, ethyl acetate, benzene, toulene,xylene(s), ethylene glycol, dichloromethane, 1,2-dichloroethane,N-methylformamide, N,N-dimethylformamide, N-methylacetamide, pyridine,dioxane, and diethyl ether. Hydrates are solvates in which the cosolventis water. It is to be understood that the definition of the compound ofthe present invention encompasses all possible hydrates and solvates, inany proportion, which possess the stated activity.

The inventors have unexpectedly discovered a novel class of P2Y₆receptor agonists, which in contrast with currently available natural orsynthetic agonists, are more metabolically stable and more selective,particularly lacking of P2Y₂ and P2Y₄ receptor activity. In general,stimulation of P2Y₆ receptors do not cause an increase of mucin releaseas does stimulation of P2Y₂ receptors (Conway J D, Am J Physiol. LungCell Mol. Physiol. 284:L945-L954 (2003)). Applicants have discoveredthat certain selective P2Y₆ receptor agonists are particularly useful inthe treatment of diseases or conditions associated with impaired ordeficient airway function, where increased airway mucus productionalready exists and the enhancement of mucociliary clearance is desired.

This invention is directed to a method of enhancing or facilitating theclearance or removal of the lung mucus secretions of a subject in needof such treatment. Lung mucus clearance depends on an optimal balance offluid, ions, surfactant and mucus secreted by the airways. Thesecomponents of lung secretions are organized in the surface of theairways as a bilayer consisting of a top mucus layer floating on thesurface of a periciliary liquid layer of approximately 7 μm thick thatallows efficient cilia movement to propel the mucus layer from the lowerrespiratory tract to the pharynx. The mucus layer protects theepithelium from inhaled pathogens and other foreign material and fromloss of fluid. The excess of mucus, the lack of fluid, and/or animbalance of ions in the airway are hallmark characteristics associatedwith patients with lung diseases that contribute to compromisedclearance of the lung mucus secretions, to recurrent pulmonaryinfections and to an overall decline in lung function associated withincreases in morbidity and mortality. These patients benefit from thetreatment with P2Y₆ agonists to promote clearance of the lungs withoutenhancing a mucus secretion.

This invention is also directed to a method of facilitating thehydration of lung mucus secretions of a subject in need of suchtreatment. This invention is further directed to a method of treatingdiseases or conditions associated within impaired or deficient airwayfunction; such diseases include chronic obstructive pulmonary disease(COPD), chronic bronchitis, emphysema, chronic asthma, chronicbronchiolitis, cystic fibrosis, primary ciliary dyskinesia (PCD), andalpha 1 antitrypsin deficiency.

The method comprises administering to a subject a therapeuticallyeffective amount of a selective P2Y₆ receptor agonist compound, whereinsaid amount is effective to bind to the P2Y₆ receptors on the luminalsurface of airway epithelia to cause mucociliary clearance; preferablywithout concomitant mucin release. The compound is effective tostimulate chloride and water secretion and therefore affects an increasein the lung hydration state, with a concomitant increase in ciliary beatfrequency.

Selective P2Y₆ Receptor Agonist Compounds

The selective P2Y₆ receptor agonist compounds useful for this inventionhave selectivity of P2Y₆ receptor over P2Y₁, P2Y₂, or P2Y₄ receptor. Theselective P2Y₆ receptor agonist compounds useful for this inventioninclude compounds of general Formula I, or salts, solvates, hydratesthereof:

wherein:

B is a purine or a pyrimidine residue according to general Formulae IVor V;

n and p=0 or 1; such that the sum of n+p is from 0 to 2, and preferably1 to 2; with the proviso that when A=M, the sum of n+p is 1;

X₁ and X₂ are independently O, NH, CH₂, CHF, CHCl, CF₂, or CCl₂;

T₁, V, and W are independently O or S;

M=H⁺, NH₄ ⁺, Na⁺ or other pharmaceutically-acceptable inorganic ororganic counter ion;

Y=OR₁;

Z=OR₂;

A=M,or

A is a nucleoside residue which is defined as:

which is linked to the phosphate chain via the 5′ position of thefuranose or carbocycle; wherein: D=O or CH₂;

Z′=H or OH

Y′=H or OH;

B′ is a purine or a pyrimidine residue according to general Formulae IVor V which is linked to the 1′-position of the furanose or carbocyclevia the 9- or 1-position of the base, respectively.

R₁ and R₂ are residues that are linked directly to the 2′- and/or3′-hydroxyls of the furanose via a carbon atom according to Formula II,or linked directly to two (2′- and 3′-) hydroxyls of the furanose via acommon carbon atom according to Formula III, such that two independentresidues of R₁ and R₂ falling within the definition of Formula II arepresent, or a combination of two independent residues made up of R₁+R₂falling within the definition of Formula III are present;

wherein:

O is the corresponding 2′- and/or 3′-oxygen of the furanose;

C is a carbon atom;

R₅, R₆, and R₇ are H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,aralkyl, aryl, substituted aralkyl, or substituted aryl, such that themoiety defined according to Formula II is an ether; or

R₅ and R₆ are H, an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,aralkyl, aryl, substituted aralkyl, or substituted aryl, and R₇ isalkoxy, cycloalkoxy, aralkyloxy, aryloxy, substituted aralkyloxy, orsubstituted aryloxy such that the moiety defined according to Formula IIis an acyclic acetal or ketal; or

R₅ and R₆ are taken together as oxygen or sulfur doubly bonded to C, andR₇ is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl,substituted aralkyl, or substituted aryl, such that the moiety definedaccording to Formula II is an ester or thioester; or

R₅ and R₆ are taken together as oxygen or sulfur doubly bonded to C, andR₇ is amino or mono- or disubstituted amino, where the substituents arealkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl,substituted aralkyl, or substituted aryl, such that the moiety accordingto Formula II is a carbamate or thiocarbamate; or

R₅ and R₆ are taken together as oxygen or sulfur doubly bonded to C, andR₇ is alkoxy, cycloalkoxy, aralkyloxy, aryloxy, substituted aralkyloxy,or substituted aryloxy, such that the moiety according to Formula II isa carbonate or thiocarbonate; or

R₇ is not present and R₅ and R₆ are taken together as oxygen or sulfurdoubly bonded to C and both the 2′- and 3′-oxygens of the furanose aredirectly bound to C to form a cyclical carbonate or thiocarbonate;

wherein:

the O atoms are the 2′- and 3′-oxygens of a furanose; and the 2′- and3′-oxygens of the furanose are linked by a common carbon atom (C) toform a cyclical acetal, cyclical ketal, or cyclical orthoester;

for cyclical acetals and ketals, R₈ and R₉ are independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl,substituted aralkyl, substituted aryl, or can be joined together to forma homocyclic or heterocyclic ring composed of 3 to 8 atoms, preferably 3to 6 atoms;

for cyclical orthoesters, R₈ is hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aralkyl, aryl, substituted aralkyl, orsubstituted aryl, R₉ is alkyloxy, cycloalkyloxy, aralkyloxy, aryloxy,substituted aralkyloxy, or substituted aryloxy;

B and B′ are independently a purine residue, as in Formula IV, linkedthrough the 9-position, or a pyrimidine residue, as in Formula V, linkedthrough the 1-position;

wherein:

R₁₀ and R₁₃ independently are hydroxy, oxo, amino, mercapto, alkylthio,alkyloxy, aryloxy, alkylamino, cycloalkylamino, aralkylamino, arylamino,diaralkylamino, diarylamino, or dialkylamino, where the alkyl groups areoptionally linked to form a heterocycle; or

R₁₀ and R₁₃ independently are acylamino according to Formula VI;

when R₁₀ in a purine or R₁₃ in a pyrimidine has as its first atomnitrogen, R₁₀ and R₁₁ or R₁₃ and R₁₄ can be taken together to form a5-membered fused imidazole ring (to give an etheno compound), optionallysubstituted on the etheno ring with one or more alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aralkyl, or aryl moieties, as described forR₅-R₉ above;

R₁₁ is hydrogen, O (adenine 1-oxide derivatives) or is absent (adeninederivatives);

R₁₄ is hydrogen, or acyl (e.g. acetyl, benzoyl, phenylacyl, with orwithout substituents);

R₁₂ is hydrogen, chlorine, amino, monosubstituted amino, disubstitutedamino, alkylthio, arylthio, or aralkylthio, where the substituent onsulfur contains up to a maximum of 20 carbon atoms, with or withoutunsaturation, and with or without substituents on the chain;

R₁₅ is hydrogen, methyl, alkyl, halogen, alkyl, alkenyl, substitutedalkenyl, alkynyl, or substituted alkynyl;

wherein:

NH is the amino residue at the C-6 position in a purine or the aminoresidue at the C-4 position in a pyrimidine;

C is a carbon atom;

W₁ is oxygen or sulfur;

R₁₆ is amino or mono- or disubstituted amino, with the aminosubstituent(s) being alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,aralkyl, or aryl, with or without further substituents, unsaturation, orheteroatoms, such that the moiety according to Formula VI is a urea orthiourea; or

R₁₆ is alkoxy, aralkyloxy, aryloxy, substituted aralkyloxy, orsubstituted aryloxy, such that the moiety according to Formula VI is acarbamate or thiocarbamate; or

R₁₆ is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, oraryl, with or without substituents or heteroatoms, such that the moietyaccording to Formula VI is an amide; with definitions of alkyl,cycloalkyl, aralkyl, or aryl groups as previously defined for comparablegroups in R₅ to R₉.

When R₅, R₆ and R₇ are not the same, or when R₈ and R₉ are not the same,a compound according to Formula I can exist in several diastereomericforms. The general structure of Formula I includes all diastereomericforms of such materials, when not specified otherwise. Formula I alsoincludes mixtures of compounds of Formula I, including mixtures ofenantiomers, diastereomers and/or other isomers in any proportion.

The ribosyl moieties in Formula I are in the D-configuration as shown,but can also be L-, or D- and L-. The D-configuration is preferred forribosyl moieties.

The Formula I compounds have the features of: (a) being a mononucleoside5′-diphosphates, dinucleoside monophosphate, dinucleoside diphosphates,or dinucleoside triphosphates; (b) both Y+OR₁ and Z═OR₂; and (c) Y′═H orOH, and Z′═H or OH. When the compound is a dinucleotide, thepolyphosphate linking the two nucleosides cannot be tetraphosphates,pentaphosphates, hexaphosphates, etc, such that the compound does nothave a significant activity toward P2Y₂ or P2Y₄ receptors. When thecompound is a dinucleotide, one nucleoside must have both Y and Zsubstituted, that is, Y and Z cannot be OH or H but must be OR; and theother nucleoside must not have either Y′ or Z′ substituted, that is Y′and Z′ must be either OH or H, such that the compound retains theactivity toward P2Y₆ receptors. It is those features that make theFormula I compounds have the selectivity of P2Y₆ receptor over other P2Yreceptors.

A preferred Formula I is Formula Ia,

wherein B₂ and B₃ are independently uracil, N-methyluracil,5-methyluracil, 5-bromouracil, 5-chlorouracil, 4-thiouracil,4-thiomethyluracil, cytosine, 5-methylcytosine, 5-bromocytosine,N-methylcytosine, N-phenylcytosine, N-benzylcytosine,N,N-dimethylcytosine, or guanosine; and

Q is phenyl, substituted phenyl, benzyl, substituted benzyl,phenylacetylene, substituted phenylacetylene, styryl, substitutedstyryl, phenethyl, or substituted phenethyl. Formula Ia also includesmixtures of compounds of Formula Ia, including mixtures of enantiomers,diastereomers and/or other isomers in any proportion.

In one embodiment, the P2Y₆ receptor agonist compounds areP¹-(2′,3′-benzylacetal uridine 5′-) P³-(uridine 5′-) triphosphate;P¹-(2′,3′-benzylacetal uridine 5′-) P³-(cytidine 5′) triphosphate; andP¹-(2′,3′-benzylacetal guanosine 5′-) P³-(uridine 5′-) triphosphate.

In another embodiment, the P2Y₆ receptor agonist compoundsP¹-(2′,3′-phenylacetal uridine 5′-) P³-(uridine 5′-) triphosphate;P¹-[2′,3′-(phenylacetylene)acetal uridine 5′-]P³-(uridine 5′-)triphosphate; and P¹-(2′,3′-phenylacetal cytidine 5′-) P³-(uridine 5′-)triphosphate.

A preferred P2Y₆ receptor agonist compound is P¹-(2′,3′-benzylacetaluridine 5′-) P³-(uridine 5′-) triphosphate. The structure is shown asbelow.

Pharmaceutical Formulation

The present invention additionally provides a pharmaceutical formulationcomprising compounds of Formula I and a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers can be selected by thoseskilled in the art using conventional criteria. Pharmaceuticallyacceptable carriers include, but are not limited to, saline solution,aqueous electrolyte solutions, isotonicy modifiers, water polyetherssuch as polyethylene glycol, polyvinyls such as polyvinyl alcohol andpovidone, cellulose derivatives such as methylcellulose andhydroxypropyl methylcellulose, polymers of acrylic acid such ascarboxypolymethylene gel, polysaccharides such as dextrans, andglycosaminoglycans such as sodium hyaluronate and salts such as sodiumchloride and potassium chloride.

The pharmaceutical formulation of the present invention provides anaqueous solution comprising water, suitable ionic or non-ionic tonicitymodifiers, suitable buffering agents, and a compound of Formula I. Inone embodiment, the compound is at 0.005 to 10% w/v, preferably 0.01-6%w/v, and the aqueous solution has a tonicity of 200-400 mOsm/kG and a pHof 4-9.

The pharmaceutical formulation can be sterilized by filtering theformulation through a sterilizing grade filter, preferably of a0.22-micron nominal pore size. The pharmaceutical formulation can alsobe sterilized by terminal sterilization using one or more sterilizationtechniques including but not limited to a thermal process, such as anautoclaving process, or a radiation sterilization process, or usingpulsed light to produce a sterile formulation. In one embodiment, thepharmaceutical formulation is a concentrated solution of the activeingredient; the formulation can be serially diluted using appropriateacceptable sterile diluents prior to systemic administration.

In one embodiment, the tonicity modifier is ionic such as NaCl, forexample, in the amount of 0.5-0.9% w/v, preferably 0.6-0.9% w/v.

In another embodiment, the tonicity modifier is non-ionic, such asmannitol, dextrose, in the amount of at least 2%, or at least 2.5%, orat least 3%, and no more than 7.5%; for example, in the range of 3-5%,preferably 3.5-5%, and more preferably 4.2-5% w/v.

The Formula I compounds can also be prepared into dry powder orequivalent inhalation powders using the well known art of super criticalfluid technology. In such a case, the Formula I compounds is admixedwith appropriate excipients and milled into a homogenous mass usingsuitable solvents or adjuvants. Following this, this mass is subjectedto mixing using super critical fluid technology and suitable particlesize distribution is achieved. The particles in the formulation need tobe of a desired particle size range such that the particles can bedirectly inhaled into the lungs using a suitable inhalation technique orintroduced into the lungs via a mechanical ventilator. Alternatively, aformulation can be designed such that the particles are large enough insize thereby offering sufficient surface area to dissolve completely ina suitable fluid when admixed together or to dissolve sufficientlyenough prior to nebulization into the lungs.

In an attempt to prevent particle size growth and minimize crystalgrowth of the Formula I compounds, one embodiment is to include the useof spray-dried particles that have better aerodynamic properties thanmicronized material. This can be further extended to coat the surface ofthe hydrophilic molecule with one or more layers of hydrophobicmaterial.

Another embodiment of this composition involves the preparation offreeze-dried or lyophilized preparation of the Formula I compounds. Thelyophilized preparations can be used as is in the form of a dry powderinhaler or be admixed with other suitable adjuvants to be used as drypowder inhaler or as a nebulized preparation.

Routes of Administration

Any method of delivering the Formula I compound to the lumen of thelung, including local administration and systemic administration, issuitable for the present invention.

A preferred embodiment of the invention is localized administration.Local administration includes inhalation, topical application ortargeted drug delivery. Methods of inhalation include liquidinstillation, instillation as a pressurized fluid preparation viametered dose inhaler or equivalent, or inhalation of an aerosolizedsolution via nebulizer (preferred), inhalation of dry powder (morepreferred), and directing soluble or dried material into the air streamduring mechanical ventilation (also more preferred).

An example of targeted drug delivery is enclosure of the Formula Icompound within a liposome, where the liposome is coated with a specificantibody whose antigen is expressed in the targeted lung tissue.Alternatively, the liposomal preparation can be designed in such afashion wherein the core of the liposome contains the Formula I compoundand the external surface of the liposomal preparation is comprised ofcationically charged moieties which traverse the alveolar surface andbind to the alveolar surface.

It can be advantageous to construe a controlled delivery system of theFormula I compound since such an inhaled product targets the site ofaction, presents the compound of interest in small regimented quantitiesand reduces/minimizes any unwanted side effects. There are somelimitations to the effectiveness of controlled release delivery systemssince prolonged residence of some of the materials in the lungs can poselong term toxicity problems.

Another example of a delivery system includes nanoparticulate ormicroparticulate compositions of the Formula I compound. In such a case,the Formula I compound is formulated as a nanosuspension wherein thecarrier is loaded with the Formula I compound; such a preparation isthen filtered through a fine porous membrane or suitable filteringmedium or is exposed to solvent interchanges to produce nanoparticles.Such nanoparticulate preparations can be freeze dried or held insuspension in an aqueous or physiologically compatible medium. Thepreparation so obtained can be inhaled by suitable means.

Another example of a suitable preparation includes a reconstitutablepreparation. In this case, the Formula I compound is formulated in apreparation to contain the necessary adjuvant to make it physiologicallycompatible. Such a preparation can be reconstituted by addition of waterfor injection or suitable physiological fluids, admixed by simpleagitation and inhaled using appropriate techniques described above.

This inhalation method involves an aerosol suspension of respirableparticles comprising the active compound, which the subject inhales. Therespirable particles can be liquid or solid, with a particle sizesufficiently small to pass through the mouth and larynx upon inhalation.In general, particles having a size of about 1 to 10 microns, preferably1-5 microns, are considered respirable.

For inhaled administration, surface concentrations of active compoundsdelivered can vary according to compounds; but are generally1×10⁻¹⁰−1×10⁻⁴ moles/liter, and preferably 1×10⁻⁸−1×10⁻⁵ moles/liter.

The active compounds can also be administered systemically to targetsites in a subject in need such that the extracellular concentration ofa P2Y₆ agonist is sufficient to elicit chloride and water secretion anda stimulation of ciliary beat frequency. The term systemic as usedherein includes subcutaneous injection, intravenous, intramuscular,intrasternal injection, intravitreal injection, infusion, inhalation,transdermal administration, oral administration, rectal administrationand intra-operative instillation.

For systemic administration such as injection and infusion, thepharmaceutical formulation is prepared in a sterile medium. The activeingredient, depending on the vehicle and concentration used, can eitherbe suspended or dissolved in the vehicle. Adjuvants such as localanesthetics, preservatives and buffering agents can also be dissolved inthe vehicle. The sterile injectable preparation can be a sterileinjectable solution or suspension in a non-toxic acceptable diluent orsolvent. Among the acceptable vehicles and solvents that can be employedare sterile water, saline solution, or Ringer's solution.

Another method of systemic administration of the active compoundinvolves oral administration, in which pharmaceutical compositionscontaining active compounds are in the form of tablets, lozenges,aqueous or oily suspensions, viscous gels, chewable gums, dispersiblepowders or granules, emulsion, hard or soft capsules, or syrups orelixirs.

For oral use, an aqueous suspension is prepared by addition of water todispersible powders and granules with a dispersing or wetting agent,suspending agent one or more preservatives, and other excipients.Suspending agents include, for example, sodium carboxymethylcellulose,methylcellulose and sodium alginate. Dispersing or wetting agentsinclude naturally-occurring phosphatides, condensation products of anallylene oxide with fatty acids, condensation products of ethylene oxidewith long chain aliphatic alcohols, condensation products of ethyleneoxide with partial esters from fatty acids and a hexitol, andcondensation products of ethylene oxide with partial esters derived fromfatty acids and hexitol anydrides. Preservatives include, for example,ethyl, and n-propyl p-hydroxybenzoate. Other excipients includesweetening agents (e.g., sucrose, saccharin), flavoring agents andcoloring agents. Those skilled in the art will recognize the manyspecific excipients and wetting agents encompassed by the generaldescription above.

For oral application, tablets are prepared by mixing the active compoundwith nontoxic pharmaceutically acceptable excipients suitable for themanufacture of tablets. These excipients can be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for example,starch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed. Formulations fororal use can also be presented as hard gelatin capsules wherein theactive ingredient is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, peanut oil, liquid paraffin or olive oil.Formulation for oral use can also be presented as chewable gums byembedding the active ingredient in gums so that the active ingredient isslowly released upon chewing.

Additional means of systemic administration of the active compound tothe lungs of the subject would involve a suppository form of the activecompound, such that a therapeutically effective amount of the compoundreaches the target sites via systemic absorption and circulation.

For rectal administration, the compositions in the form of suppositoriescan be prepared by mixing the active ingredient with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the compound. Such excipients include cocoa butter andpolyethylene glycols.

The active compounds can also be systemically administered to the lungsof the subject through absorption by the skin using transdermal patchesor pads. The active compounds are absorbed into the bloodstream throughthe skin. Plasma concentration of the active compounds can be controlledby using patches containing different concentrations of activecompounds.

One systemic method involves an aerosol suspension of respirableparticles comprising the active compound, which the subject inhales. Theactive compound would be absorbed into the bloodstream via the lungs.The respirable particles can be liquid or solid, with a particle sizesufficiently small to pass through the mouth and larynx upon inhalation;in general, particles ranging from about 1 to 10 microns, but morepreferably 1-5 microns, in size are considered respirable.

Another method of systemically administering the active compounds to thelungs of the subject involves administering a liquid or a liquidsuspension in the form of eye drops or eye wash or nasal drops of aliquid formulation, or a nasal spray of respirable particles that thesubject inhales. Liquid pharmaceutical compositions of the activecompound for producing a nasal spray or nasal or eye drops can beprepared by combining the active compound with a suitable vehicle, suchas sterile pyrogen free water or sterile saline by techniques known tothose skilled in the art.

For systemic administration, plasma concentrations of active compoundsdelivered can vary according to compounds; but are generally1×10⁻¹⁰−1×10⁻⁴ moles/liter, and preferably 1×10⁻⁸−1×10⁻⁵ moles/liter.

Packaging

The selective P2Y₆ agonist compound can be packaged so as to allow apractitioner to formulate it into pharmaceutical compositions as needed.Alternatively, the pharmaceutical composition, itself, can be packaged,thereby requiring de minimus formulation by practitioner. In any event,the packaging should maintain efficacy and chemical and aestheticintegrity of therapeutic material or pharmaceutical composition to theextent reasonably possible.

Possible methods of packaging include blister packaging, packaging inunit dose vials, packaging in blow-fill-seal plastic vials, filling inpressurized canisters, packaging in a two compartment system wherein thecontents of the two compartments are admixed by mechanical agitationprior to administration and the contents used within a specified periodof time. Packaging the material by filling into a plastic vial whosecontents can be easily opened, contents dispensed, and empty containerdisposed off to prevent re-use or contamination is deemed advantageous.The most preferred packaging method is packaging the formulation in ablister packaging system wherein the contents are held protected fromheat, light, and other environmental extremes.

Where the therapeutic material is packaged for inhalation, thepharmaceutical composition can be packaged in aerosol spray canister orpackaged for use with nebulizer or ventilator. This can be achieved bydirectly filling the container using the common techniques of coldfilling or filling under a pressurized system or simply filling theproduct formulation under gravity feed in an aseptic environment.Depending on the nature of the final formulation, this can be achievedby a cold-filling technique wherein the composition is packaged in anaerosol canister under high pressure in a clean-room environment,preferably under aseptic conditions. Alternatively, if the compositionis a simple solution, homogenous fluid, or well-mixed suspensionproduct, the formulation can be filled into unit dose blow-fill-sealvials under a gravity feed or filled into blister packs wherein theformulation is filled into unit cavities and secured close with suitablefoil or equivalent packaging to protect it from environmental extremes.This operation is preferred to be carried out under aseptic conditions,preferably under ambient or sub-ambient temperatures with little to noenvironmental extremes. It is desirable that such filling and packagingoperations be conducted in relatively particulate free environments withminimal microbiological loads (especially absence of Pseudomonas andother similar pathogens) and be done with minimal exposure to directhuman interface. The blister packaging can be done most optimally withcold fill packaging. The product compositions can be directly filledinto the final container of choice by direct metered transfer (eithergravimetrically or volumetrically) and secured close with appropriateclosure systems.

The invention is illustrated further by the following examples that arenot to be construed as limiting the invention in scope to the specificprocedures described in them.

EXAMPLES Example 1 Preparation of P¹-(2′,3′-benzylacetal uridine 5′-)P³-(uridine 5′-) triphosphate, Ammonium Salt

Uridine 5′-diphosphate, tributylammonium salt (UDP.NBu₃)

Uridine 5′-diphosphate, disodium salt (UDP.2Na) was converted to thefree acid by stirring with Dowex 50 H⁺ or equivalent (5 g resin/gUDP.2Na) in water for 10 minutes. The resin was filtered and thefiltrate was combined with tributylamine (1.5 eq.). The mixture wasstirred vigorously for 15 minutes, such that the pH of the aqueous layerremained above 8. The solution was evaporated at <35° C., and theresidue co-evaporated with dry N,N-dimethylformamide (3×), at <40° C.The residue was lyophilized overnight to a dry glassy foam.

Uridine 5′-monophosphate, tributylammonium salt (UMP.NBu₃)

Uridine 5′- monophosphate, free acid (UMP) was converted to themono-tributylammonium salt by treatment with tributylamine (1.5 eq.) inwater. Following solvent removal and co-evaporation with dryN,N-dimethylformamide, the product was lyophilized overnight to aconstant weight.

P¹, P³-di(uridine 5′-) triphosphate, triammonium salt

UMP.NBu₃ (380 mg, 0.747 mmol, 1.1 eq) was dissolved in dryN,N-dimethylformamide (2 mL) and 1,1′-carbonyldiimidazole (242 mg, 1.49mmol, 2.2 eq) added in a single portion. The solution was stirred atroom temperature for 1 hour. ³¹P NMR indicated almost completeconversion to the imidazolide, and methanol (55 uL, 2 eq) was added toquench the excess of CDI. After 30 minutes stirring, UDP.NBu₃ (400 mg,0.679 mmol, 1 eq) was added as a solid. The reaction mixture was stirredat 45° C. for 24 hours, with HPLC monitoring (Hamilton PRP-X100 column,250×4.1 mm, 10 um, gradient from water to 90% 1 M NH₄HCO₃/10% ACN over30 or 60 minutes, 2 mL/min, monitor at 260 nm). At the end of thereaction HPLC indicated that the desired product constituted about 45%of the total nucleotide content. Water (500 uL) was added to quench anyresidual activated species and the solvents were evaporated at <40° C.and <5 mm Hg. The residue was reconstituted in water and the product wasisolated by preparative HPLC. (Hamilton PRP-X100 column, 250×50 mm,12-20 um, gradient from water to 90% 1 M NH₄HCO₃/10% ACN over 30minutes; 100 mL/min, monitor at 260 nm). The yield of the title compoundwas 140 mg (27%).

P¹-(2′,3′-benzylacetal uridine 5′-) P³-(uridine 5′-) triphosphate,ammonium salt

P¹, P³-(uridine 5′-) triphosphate, triammonium salt (1.0 g, 1.31 mmol, 1eq) was dissolved in 98% formic acid (5 mL) and phenylacetaldehyde,dimethylacetal (0.435 mL, 2.62 mmol, 2 eq) was added. The reactionmixture was stirred for three days at room temperature, at which pointHPLC (C₁₈) indicated a 43% conversion to the desired monoacetal product,along with 14% of the undesired diacetal. The formic acid was removed aswell as possible by evaporation, and the residue neutralized to pH 8with 1 M sodium bicarbonate (30 mL). The aqueous solution was washedwith ethyl acetate (2×20 mL) to remove excess aldehyde, and concentratedto 12 mL. The product was isolated via preparative HPLC (NovaPak C₁₈column, 25×200 mm, gradient from 0.1 M ammonium acetate (pH 5.9) tomethanol over 25 minutes, monitor at 260 nm). The yield of the titlecompound was 0.533 g. (47%).

¹H NMR (D₂O, 300 MHz) δ: 2.93 (d, 2H), 3.93-4.20 (m, 8H), 4.65 (m, 2H),15.23 (t, 1H), 5.31 (d, 1H), 5.73 (m, 3H), 7.16 (m, 5H), 7.54 (d, 1H),7.73 (d, 1H). ³¹P NMR (D₂O, 121.47 MHz): −10.30 (d, 1P), −10.82 (d, 1P),−21.99 (m, 1P). MW calculated for C₂₆H₃₁N₄O₂₀P₃ (MH⁻) 811, found 811.3by LCMS.

Example 2 Selectivity of UDP, UP₃U and Mono-benzylacetal UP₃U

Human astrocytoma (1321N1) cells expressing P2Y₁, P2Y₂, P2Y₄, and P2Y₆were grown to confluency in 96-well plates. Cells were loaded with asolution of Fluo-3 AM (2.5 μM final concentration) in an assay bufferconsisting of 10 mM KCl, 118 mM NaCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 20 mMHEPES, 10 mM glucose, pH 7.4. After a 60-minute incubation with Fluo-3AM at 25° C., cells were washed and stimulated with serially dilutedconcentrations of compounds uridine 5′-diphosphate (UDP), P¹,P³-(diuridine 5′-) triphosphate (UP₃U) or P¹-(2′,3′-benzylacetal uridine5′-) P³-(uridine 5′-) triphosphate (mono-benzylacetal UP₃U).Intracellular calcium levels were monitored in each well simultaneouslyby measuring the changes in fluorescence intensity using the FLIPR(Molecular Devices Corp., Sunnyvale, Calif.). The results of this assayare presented in Table 1. Values for compounds UDP, UP₃U, andmono-benzylacetal UP₃U in Table 1 are presented as EC₅₀ valuescorresponding to the concentration of an agonist that produces 50% ofthe maximum possible response. Since mono-benzylacetal UP₃U has activityonly at P2Y₆ receptors and requires the lowest concentration to elicit aresponse, these results illustrate that mono-benzylacetal UP₃U is apotent and selective agonist of P2Y₆ receptors. In a separateexperiment, di-benzylacetal UP₃U (an analog of mono-benzylacetal UP₃U,where the two remaining hydroxyl groups are also substituted with abenzylacetal moiety, such that Y, Y′, Z, and Z′ are all OR), showed noresponse at P2Y₆.

TABLE 1 Potency and P2Y receptor selectivity. EC₅₀ (μM) Compound P2Y₁P2Y₂ P2Y₄ P2Y₆ UDP NR 4.20 9.48 0.50 UP₃U NR 13 SR 0.92 mono- NR NR NR0.14 benzylacetal UP₃U NR = No response SR = Small response at 100 μM

Example 3 Chloride Secretion in Human Nasal Airway Cells

Induction of chloride secretion in vivo facilitates hydration ofthickened airway mucus secretions in diseases where patients willbenefit from mobilization and clearance of such secretions. Activationof an apical non-CFTR chloride channel induce efflux of chloride ionsand water that help rehydrate the lung secretions (Boucher, U.S. Pat.No. 5,292,498 and Boucher, U.S. Pat. No. 5,635,160 and referencestherein).

Airway epithelial cells are dispersed and isolated from freshly excisedhuman nasal surgical specimens (Yankaskas, et al., Am Rev Resp Dis 132,1281-1287 (1985)). Confluent monolayers are cultured on permeablecollagen matrix supports in F-12 hormone-supplemented medium (Wu, etal., Am Rev Resp Dis 132, 311-320 (1985)). Cells are incubated at 37° C.and grown to confluence. Development of transepithelial resistance ismonitored to determine the formation of tight junctions between cells.After formation of tight junctions is confirmed, the matrix supportscontaining the cultures are mounted in modified Using chambers.

Cultured human airway epithelia are mounted in Using chambers with asubmucosal bath of Krebs Bicarbonate Ringer, (KBR (in mM), 140 Na⁺, 120Cl⁻, 5.2 K⁺, 25 HCO₃ ⁻, 2.4 HPO₄ ²⁻, 0.4 HPO₄ ⁻, 1.1 Ca² ⁺, 1.2 Mg²⁺,and 5.2 glucose, pH 7.4). The luminal surface is bathed by KBr or by ahigh K⁺, low Cl⁻ Ringers ((in mM) 40 Na⁺, 100 K⁺, 4.5 Cl⁻, 120gluconate, 25 HCO₃ ⁻, 2.4 HPO₄ ²⁻, 0.4 HPO₄ ⁻, 1.1 Ca² ⁺, 1.2 Mg²⁺, and5.2 glucose, pH 7.4).

Bioelectric properties including short-circuit current (I_(SC)),transpeithelial potential difference and resistance are monitored.I_(SC) is measured with a digital voltmeter and plotted on a strip chartrecorder. The open circuit potential is recorded periodically andconductance is monitored in the voltage clamp mode by the currentdeflection in response to a 10 mV voltage pulse. An increase in I_(SC)is a measure of the activation of ion transport that leads to fluidtransport and enhancement of mucocilliary clearance of the airways.

A stable baseline of I_(SC) is recorded and amiloride (100 μM) is addedto the solution bathing the apical surface to block the sodiumabsorption. The residual I_(SC) measured under these conditions is agood approximation to chloride secretion (Boucher, et al., J. Clin.Invest. 78: 1245 (1986); Willumsen, et al., Am J Physiol 256: C226-C233;C1033-C1044; C1045-C1053 (1989)). After recording a stable baseline, asolution of a selective P2Y₆ agonist compound such as mono-benzylacetalUP₃U is added to the chamber bathing the apical surface of theepithelial culture. The change in I_(SC) is recorded.Concentration-response curves are obtained by cumulative addition ofhigher concentrations of test compound in 0.5 log steps.

The P2Y₆ agonist compound increases the short circuit current (I_(SC))in a dose-dependent manner, demonstrating an increase in chloridesecretion and water, thus promoting the hydration of the airways.

Example 4 Ciliary Beat Frequency

The effects of a selective P2Y₆ agonist compound such asmono-benzylacetal UP₃U on ciliary activity are determined on individualhuman ciliated nasal epithelial cells using techniques describedpreviously (Geary et al., Am J Physiol. 268, L1021-8 (1995); Morse etal. Am. J. Physiol. Cell Physiol. 280:C1485-497 (2001)). Briefly,epithelial cells are recovered from protease digests of human nasalturbinates, obtained from normal subjects and from patients with lungdiseases such as cystic fibrosis, chronic bronchitis, primary ciliarydyskinesia and COPD. The cells are seeded into 12-mm CostarTranswell-Col cell culture supports at a density of 300,000 cells/cm²and incubated overnight in hormone-supplemented culture medium (Gray etal., Am J Respir Cell Mol Biol 14: 104-112 (1996)) at 37° C. in anatmosphere of air (5% CO₂), after which nonadherent cells are washedaway to reveal small explants of the superficial epithelium as smallclumps of ciliated cells that have attached to the substratum. Thesepreparations are used within 4 days. Transwell-Col cell culture supportsbearing epithelial explants are mounted in a chamber on the stage of aninverted microscope, superfused luminally, and warmed (35° C.) asdescribed previously (Morse et al., 2001). The control superfusion andthe serosal bathing solution is Krebs-Ringer bicarbonate (KRB) with thefollowing composition: 125 mM NaCl, 5.2 mM KCl, 1.2 mM MgCl₂, 1.2 mMCaCl₂, 25 mM NaHCO₃, 10 mM TES, 5 mM glucose (pH 7.4 when gassed with 5%CO₂). The explanted, native ciliated cells are viewed by phase contrastmicroscopy using a Zeiss IM microscope (Carl Zeiss Inc., Thornwood,N.Y.) and 32× objective, and the image is monitored with a Dage 72monochrome charge-coupled device video camera (Dage-MTI, Michigan City,Ind.). Ciliary beat frequency (CBF) is determined using a photosensorpositioned over the image of an individual cell on the face of the videomonitor to detect ciliary beating, as previously described (Morse etal., 2001). In all experiments, cultures are equilibrated with 1.5 h ofsuperfusion with KRB. Each preparation is then subjected to two 10-minbaseline and agonist stimulation periods, with variable concentrationsof test compound, then with 100 μM UDP. Data are recorded every minutefor the determination of CBF. A 30-min KRB washout period separated thetest compound challenge from a subsequent second baseline period. Afterfast Fourier transformation analyses for each experiment, the resultingCBF data are normalized to the respective mean baseline CBF. For thetest compound concentration-response study, the peak CBF is obtained andcompared with that obtained with 100 μM UDP. The data are reported asthe mean±S.E. of the peak response, relative to baseline. Culturesderived from the tissues of three or more patients are used.

The P2Y₆ agonist compound increases in a dose-dependent manner the beatfrequency of cilia isolated from normal subjects and from patients withlung diseases such as cystic fibrosis, chronic bronchitis, primaryciliary dyskinesia and COPD, demonstrating improvements on mucociliaryclearance.

Example 5 Mucin Secretion

Primary normal human tracheal/bronchial epithelial cells(donor-specific, nonsmoker), which are shipped cryopreserved in thepresence of retinoic acid, are obtained from Clonetics (East Rutherford,N.J.; CC-2540). The cells are initially seeded on Transwell-Clearculture inserts (Corning-Costar 3460; Corning) and grown in bronchialepithelial growth medium (BEGM) (Clonetics; CC-3170 BEGM BulletKit basemedia, plus supplements). After 2 to 3 days in culture, cells areswitched to air/liquid interface (ALI) culture conditions as has beenpreviously described by Gray et al. (1996). The 17Q2 mucin antibodypurified with a Protein G column (Pierce, Rockford, Ill.) from ascitesfluid (University of California at Davis) is conjugated to alkalinephosphatase using the EZ-Link maleimide-activated alkaline phosphatasekit (Pierce).

Known amounts of a selective P2Y₆ agonist compound such asmono-benzylacetal UP₃U are added to the apical surface of the culturesand incubated at 37° C. for 2 h. At the end of incubation period,mucin-containing cell supernatants are removed from the apicalcompartment and stored at −70° C. Estimation of mucin production iscarried out using an antigen/antibody enzyme-linked immunoassay asdescribed previously (Wright et al., Am J Physiol 271: L854-L861(1996)).

Example 6 Tracheal Mucus Velocity

Prior to the study, approval of the Animal Research Committee, to assurethe humane care and use of experimental animals is obtained. Adult ewes,25 to 45 kg in weight, are restrained in an upright position in aspecialized body harness adapted to a modified shopping cart. The headsof the animals are immobilized, and local anesthesia of the nasalpassage is induced with 2% lidocaine. Following topical anesthesia ofthe nasal passages with 2% lidocaine solution, the sheep are nasallyintubated with an endotracheal tube 7.5 cm in diameter (MallinckrodtMedical Inc., St. Louis, Mo.), which has been shortened by 6 cm. Thecuff of the tube is placed just below the vocal cords (verified byfluoroscopy) to allow for maximal exposure of the tracheal surface area.After intubation, the animals are allowed to acclimate for a period of20 min before beginning measurements of Tracheal Mucus Velocity (TMV).During the course of the experiment, the inspired air is warmed andhumidified using a Bennett humidifier (Puritan-Bennett, Lenexa, Kans.).To minimize possible impairment of TMV caused by inflation of theendotracheal tube cuff, the cuff is deflated throughout the study,except for the period of drug delivery. The sheep are periodicallygavaged with tap water to avoid dehydration (Sabater et al., J ApplPhysiol 87: 2191-2196 (1999)). TMV is measured in vivo by aroentgenographic technique as previously described (Sabater et al., Am JRespir Crit Care Med 154: 341-345 (1996), Sabater et al., J Appl Physiol87: 2191-2196 (1999)). Between 10 and 12 radiopaque Teflon/bismuthtrioxide disks, which are 1 mm in diameter, 0.8 mm thick, and 1.8 mg inweight, are insufflated into the trachea. A modified suction catheterconnected to a source of continuous compressed air at a flow of 3 to 4l/min is used to introduce the particles via the endotracheal tube. Thecatheter is maintained within the endotracheal tube only, so that nocontact with the tracheal surface is made. The cephalad-axial velocitiesof the individual disks are recorded on videotape from a portable imageintensifier unit. Individual disk velocities are calculated by measuringthe distance traveled by each disk during a 1-min observation period.For each run, the mean value of all individual disk velocities iscalculated. A collar containing radiopaque reference markers of knownlength is worn by the sheep during the study and used as a standard tocorrect for magnification effects inherent in the fluoroscopy unit.

After obtaining a baseline TMV, 4 ml aliquots of several concentrationsof a P2Y₆ agonist compound such as mono-benzylacetal UP₃U or placebo areadministered by nebulization in sterile saline. The agents are deliveredto the animals with a Pari LC Star nebulizer (Pari Respiratory, RichmondVa.), via free breathing. The nebulizer is driven by room air at a flowrate of 8 LPM, and the time to reach dryness is approximately 10 to 12min. TMV is measured immediately after drug administration (0 h) and at0.25, 0.5, 1, 2, 4, 6, and 8 h after treatment.

The P2Y₆ agonist compound increases the velocity of the cephalad axialdisplacement of radiopaque particles from the sheep trachea in adose-dependent manner, demonstrating that the compound increasesclearance of the airways.

Example 7 Treatment of Primary Ciliary Dyskinesia

A selective P2Y₆ agonist compound such as mono-benzylacetal UP₃U isadministered to patients diagnosed with primary ciliary dyskinesia (PCD)(verified by electron microscopy analysis of ciliary ultrastructuredefect from nasal biopsy). The efficacy P2Y₆ receptor agonists isdetermined by measuring the clearance of inhaled radiolabeled particlesfrom the lung by radionuclide scanning techniques using a gamma camera.Each subject inhales an aerosol of iron oxide labeled with Technetium⁹⁹m (⁹⁹Tc—Fe₂O₃). Subjects inhale the aerosol for approximately 5minutes. Subjects are then seated in front of a gamma camera, and forthe next 20 minutes the subjects randomly inhale either a salinecontrol, or an effective concentration the P2Y₆ receptor agonist(0.1-100 mg/mL) for approximately 20 minutes. After this inhalation,subjects remain seated in front of the gamma camera for the next twohours to measure clearance of the radiolabeled iron oxide. The efficacyof the P2Y₆ receptor agonists in treating primary ciliary dyskinesia isdemonstrated by an improvement in cough clearance of the ⁹⁹TC—Fe₂O₃ ascompared to the saline vehicle alone.

Example 8 Treatment of Chronic Bronchitis

A selective P2Y₆ agonist compound such as mono-benzylacetal UP₃U isadministered to patients diagnosed with chronic bronchitis (based uponthe American Toracic Society definition: excess mucus production,occurring on most days for at least three months of the year for atleast two successive years). The efficacy of the P2Y₆ receptor agonistis determined by measuring the clearance of inhaled radiolabeledparticles from the lung by radionuclide scanning techniques using agamma camera. Each subject inhales an aerosol of iron oxide labeled withTechnetium ⁹⁹m (⁹⁹Tc—Fe₂O₃). Subjects inhale the aerosol forapproximately 5 minutes. Subjects are then seated in front of a gammacamera, and for the next 20 minutes the subjects randomly inhale eithera saline control, or an effective concentration of the P2Y₆ receptoragonist (0.1-100 mg/mL) for approximately 20 minutes. After thisinhalation, subjects remain seated in front of the gamma camera for thenext two hours to measure clearance of the radiolabeled iron oxide. Somestudies include subjects that perform controlled coughs in this timeperiod, and sputum is collected, weighed, and volume is recordedthroughout the study, and stored for additional analysis of sputumrheology or iron content. Subjects repeat this procedure on subsequentdays as appropriate to the number of doses under study. The efficacy ofthe inhaled P2Y₆ receptor agonists in treating chronic bronchitis isdemonstrated by an improvement in mucociliary and/or cough clearance ofthe ⁹⁹TC—Fe₂O₃ as compared to the saline vehicle alone.

Approximately 24 hours following each inhalation exposure, subjects aresubjected to a 30-minute scan of residual radioactivity in the lung.During this time they sit continuously in front of the gamma camera.

Safety data is collected by monitoring heart rate, ECG, blood pressure,oxyhemoglobin saturation by pulse oximetry prior to, during and afterinhalation for all dosing periods. All patients during all phases of thestudy are monitored for any adverse reactions during each dose periodbeginning with inhalation of study drug and ending after the 30 minutescanning at 24 hours.

Example 9 Treatment of Chronic Obstructive Pulmonary Disease (COPD)

A selective P2Y₆ agonist compound such as mono-benzylacetal UP₃U isadministered to patients diagnosed with Chronic Obstructive PulmonaryDisease (COPD), patients with airflow limitation that is not fullyreversible. The airflow limitation is usually both progressive andassociated with an abnormal inflammatory response of the lungs tonoxious particles or gases. Patients have symptoms of cough, sputumproduction, or dyspnea (difficult or labored breathing), and/or ahistory of exposure to risk factors for the disease. The diagnosis isconfirmed by spirometry (measurements of lung function and capacity) andclinical symptoms and signs, such as abnormal shortness of breath andincreased forced expiratory time). The efficacy the P2Y₆ receptoragonists is determined by measuring the clearance of inhaledradiolabeled particles from the lung by radionuclide scanning techniquesusing a gamma camera. Each subject inhales an aerosol of iron oxidelabeled with Technetium ⁹⁹m (⁹⁹Tc—Fe₂O₃). Subjects inhale the aerosolfor approximately 5 minutes. Subjects are then seated in front of agamma camera, and for the next 20 minutes the subjects randomly inhaleeither a saline control, or an effective concentration of the P2Y₆receptor agonist (0.1-100 mg/mL) for approximately 20 minutes. Afterthis inhalation, subjects remain seated in front of the gamma camera forthe next two hours to measure clearance of the radiolabeled iron oxide.Some studies include subjects that perform controlled coughs in thistime period, and sputum is collected, weighed, and volume is recordedthroughout the study, and stored for additional analysis of sputumrheology or iron content. Subjects repeat this procedure on subsequentdays as appropriate to the number of doses under study. The efficacy ofthe inhaled P2Y₆ receptor agonists in treating COPD is demonstrated byan improvement in mucociliary and/or cough clearance of the ⁹⁹TC—Fe₂O₃as compared to the saline vehicle alone.

Approximately 24 hours following each inhalation exposure, subjects aresubjected to a 30-minute scan of residual radioactivity in the lung.During this time they sit continuously in front of the gamma camera.

Safety data is collected by monitoring heart rate, ECG, blood pressure,oxyhemoglobin saturation by pulse oximetry prior to, during and afterinhalation for all dosing periods. All patients during all phases of thestudy are monitored for any adverse reactions during each dose periodbeginning with inhalation of study drug and ending after the 30 minutescanning at 24 hours.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications could be made without departing from the scope of theinvention.

1. A method of enhancing or facilitating the clearance of the lung mucussecretions of a subject in need of such treatment, comprisingadministering to the lungs of the subject a compound of Formula I, or apharmaceutically acceptable salt thereof, in an amount sufficient toclear mucus secretions;

wherein: B is a purine or a pyrimidine residue according to generalFormulae IV or V; n and p=0 or 1; such that the sum of n+p is from 0 to2, with the proviso that when A=M, the sum of n+p is 1; X₁ and X₂ areindependently O, NH, CH₂, CHF, CHCl, CF₂, or CCl₂; T₁, V, and W areindependently O or S; M=H⁺, NH₄ ⁺, Na⁺ or otherpharmaceutically-acceptable inorganic or organic counter ion; Y=OR₁;Z=OR₂; A=M, or A is a nucleoside residue which is defined as:

wherein: D=O; Z′=H or OH; Y′=H or OH; B′ is a purine or a pyrimidineresidue according to general Formulae IV or V which is linked to the1′-position of the furanose or carbocycle via the 9-or 1-position of thebase, respectively; R₁ and R₂ are residues that are linked directly tothe 2′- and /or 3′-hydroxyls of the furanose via a carbon atom accordingto Formula II, or linked directly to two (2′- and 3′-) hydroxyls of thefuranose via a common carbon atom according to Formula III;

wherein O is the corresponding 2′- and/or 3′-oxygen of the furanose; Cis a carbon atom; R₅, R₆, and R₇ are independently H, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl, substituted aralkyl,or substituted aryl, such that the moiety defined according to FormulaII is an ether; or R₅ and R₆ are independently H, an alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl, substituted aralkyl,or substituted aryl, and R₇ is alkoxy, cycloalkoxy, aralkyloxy, aryloxy,substituted aralkyloxy, or substituted aryloxy such that the moietydefined according to Formula II is an acyclic acetal or ketal; or R₅ andR₆ are taken together as oxygen or sulfur doubly bonded to C, and R₇ isalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl,substituted aralkyl, or substituted aryl, such that the moiety definedaccording to Formula II is an ester or thioester; or R₅ and R₆ are takentogether as oxygen or sulfur doubly bonded to C, and R₇ is amino ormono- or disubstituted amino, where the substituents are alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl, substituted aralkyl,or substituted aryl, such that the moiety according to Formula II is acarbamate or thiocarbamate; or R₅ and R₆ are taken together as oxygen orsulfur doubly bonded to C, and R₇ is alkoxy, cycloalkoxy, aralkyloxy,aryloxy, substituted aralkyloxy, or substituted aryloxy, such that themoiety according to Formula II is a carbonate or thiocarbonate; or R₇ isnot present and R₅ and R₆ are taken together as oxygen or sulfur doublybonded to C and both the 2′- and 3′-oxygens of the furanose are directlybound to C to form a cyclical carbonate or thiocarbonate;

wherein the O atoms are the 2′- and 3′-oxygens of a furanose; and the2′- and 3′-oxygens of the furanose are linked by a common carbon atom(C) to form a cyclical acetal, cyclical ketal, or cyclical orthoester;for cyclical acetals and ketals, R₈ and R₉ are independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl,substituted aralkyl, or substituted aryl, or R₈ and R₉ are joinedtogether to form a homocyclic or heterocyclic ring composed of 3 to 8atoms, for cyclical orthoesters, R₈ is hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl, substituted aralkyl,or substituted aryl, R₉ is alkyloxy, cycloalkyloxy, aralkyloxy, aryloxy,substituted aralkyloxy, or substituted aryloxy; B and B′ areindependently a purine residue, as in Formula IV, linked through the9-position, or a pyrimidine residue, as in Formula V, linked through the1-position;

wherein: R₁₀ and R₁₃ independently are hydroxy, oxo, amino, mercapto,alkylthio, alkyloxy, aryloxy, alkylamino, cycloalkylamino, aralkylamino,arylamino, diaralkylamino, diarylamino, or dialkylamino, where the alkylgroups are optionally linked to form a heterocycle; or R₁₀ and R₁₃independently are acylamino according to Formula VI; when R₁₀ in apurine or R₁₃ in a pyrimidine has as its first atom nitrogen, R₁₀ andR₁₀or R₁₃ and R₁₄ are optionally taken together to form a 5-memberedfused imidazole ring to give an etheno compound, optionally substitutedon the etheno ring with one or more alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, aralkyl, or aryl moieties, as described for R₅-R₉above;R₁₁ is hydrogen, O or is absent; R₁₄ is hydrogen, or acyl; R₁₂ ishydrogen, chlorine, amino, monosubstituted amino, disubstituted amino,alkylthio, arylthio, or aralkylthio, where the substituent on sulfurcontains up to a maximum of 20 carbon atoms, with or withoutunsaturation, and with or without substituents on the chain; R₁₅ ishydrogen, methyl, alkyl, halogen, alkyl, alkenyl, substituted alkenyl,alkynyl, or substituted alkynyl;

wherein C is a carbon atom; W₁ is oxygen or sulfur; R₁₆ is amino ormono- or disubstituted amino, such that the moiety according to FormulaVI is a urea or thiourea; or R₁₆ is alkoxy, aralkyloxy, aryloxy,substituted aralkyloxy, or substituted aryloxy, such that the moietyaccording to Formula VI is a carbamate or thiocarbamate; or R₁₆ isalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, or aryl,with or without substituents or heteroatoms, such that the moietyaccording to Formula VI is an amide.
 2. A method of facilitating thehydration of lung mucus secretions of a subject in need of suchtreatment, comprising administering to the lungs of the subject acompound of Formula I as described in claim 1, or a pharmaceuticallyacceptable salt thereof, in an amount sufficient to hydrate lungsecretions.
 3. A method of treating a disease or condition associatedwith impaired or deficient airway function, comprising administering tothe lungs of the subject a compound of Formula I as described in claim1, or a pharmaceutically acceptable salt thereof, in an amount effectiveto activate P2Y₆ receptors on airway epithelia, wherein said disease ischronic obstructive pulmonary disease, chronic bronchitis, emphysema,cystic fibrosis, primary ciliary dyskinesia, or alpha 1 antitrypsindeficiency.
 4. The method according to claim 3, wherein said disease ischronic bronchitis.
 5. The method according to claim 3, wherein saiddisease is chronic obstructive pulmonary disease.
 6. The methodaccording to claim 3, wherein said disease is primary ciliarydyskinesia.
 7. The method according to claim 3, wherein said disease iscystic fibrosis.
 8. The method according to any one of claims 1-3,wherein said compound of Formula I is a compound of Formula Ia,

wherein B₂ and B₃ are independently uracil, N-methyluracil,5-methyluracil, 5-bromouracil, 5-chlorouracil, 4-thiouracil,4-thiomethyluracil, cytosine, 5-methylcytosine, 5-bromocytosine,N-methylcytosine, N-phenylcytosine, N-benzylcytosine,N,N-dimethylcytosine or guanosine; and Q is phenyl, substituted phenyl,benzyl, substituted benzyl, phenylacetylene, substitutedphenylacetylene, styryl, substituted styryl, phenethyl, or substitutedphenethyl.
 9. The method according to claim 8, wherein said compound isselected from the group consisting of: P¹-(2′,3′-benzylacetal uridine5′-) P³-(uridine 5′-) triphosphate; P¹-(2′,3′-benzylacetal uridine 5′-)P³-(cytidine 5′) triphosphate; P¹-(2′,3′-benzylacetal guanosine 5′-)P³-(uridine 5′-) triphosphate; P¹-(2′,3′-phenylacetal uridine 5′-)P³-(uridine 5′-) triphosphate; P¹-[2′,3′-(phenylacetylene)acetal uridine5′-] P³-(uridine 5′-) triphosphate; and P¹-(2′,3′-phenylacetal cytidine5′-) P³-(uridine 5′-) triphosphate.
 10. The method according to claim 9,wherein said compound is P¹-(2′,3′-benzylacetal uridine 5′-) P³-(uridine5′-) triphosphate.
 11. The method according to claim 3, wherein saidcompound is administered by inhalation.
 12. The method according toclaim 3, wherein said compound is administered in a form of an aerosolsuspension of respirable particles.
 13. The method according to claim12, wherein said respirable particles have a size of 1-10 micron. 14.The method according to any one of claims 1-3, wherein X₁, X₂, T₁, V,and W are O.